All existing global gene expression assay techniques require relatively large quantities of analyte;in order to use them with the minute amount of mRNA present in a few or a single cell, enzymatic amplification is required . This process is time consuming, technically difficult, and expensive. More importantly, the amplification process itself biases the sample in a way that prohibits truly quantitative analysis . This, combined with specific limitations of microarrays and DNA sequencing, results in no straightforward method to study transcription in single cells. To solve this problem we aim to directly identify individual gene transcript molecules (in the form of cDNA) via atomic force microscopy.
The Specific Aims of this Application are to combine the building blocks of hardware, software and chemistry that we have developed into an integrated, functional system. The improvement we are proposing will significantly impact medicine by reducing time, cost and technical complexity of small sample transcriptional profiling;and will lower the bioinformatics burden by producing easier to interpret, better quality of information.
Project Narrative The long term goal of our research is to dissect, understand, and control the biology of single cells in complex tissues, such as brain, or in malignant tumors. Furthering this body of work requires that we address an unsolved problem in single cell molecular analysis: the lack of a method to routinely, reliably and inexpensively determine global gene transcriptional activity. The nanotechnologies we are developing will significantly impact medicine by reducing time, cost and technical complexity of small sample transcriptional profiling;and will lower the bioinformatics burden by producing easier to interpret, better quality of information.
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|Reed, Jason; Hsueh, Carlin; Lam, Miu-Ling et al. (2012) Identifying individual DNA species in a complex mixture by precisely measuring the spacing between nicking restriction enzymes with atomic force microscope. J R Soc Interface 9:2341-50|